Î±-Glycosylation by d-Glucosamine-Derived Donors: Synthesis of

Article pubs.acs.org/JACS

α-Glycosylation by D-Glucosamine-Derived Donors: Synthesis of Heparosan and Heparin Analogues That Interact with Mycobacterial Heparin-Binding Hemagglutinin Medel Manuel L. Zulueta,†,‡,# Shu-Yi Lin,†,§,# Ya-Ting Lin,§ Ching-Jui Huang,§ Chun-Chih Wang,§ Chiao-Chu Ku,† Zhonghao Shi,† Chia-Lin Chyan,*,∥ Deli Irene,∥ Liang-Hin Lim,∥ Tsung-I Tsai,† Yu-Peng Hu,†,§ Susan D. Arco,‡ Chi-Huey Wong,† and Shang-Cheng Hung†,*,⊥ †

Genomics Research Center, Academia Sinica, No. 128, Section 2, Academia Road, Taipei 115, Taiwan Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101, Philippines § Department of Chemistry, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 300, Taiwan ∥ Department of Chemistry, National Dong Hwa University, No. 1, Sec. 2, Ta-Hsueh Road, Shoufeng, Hualien 974, Taiwan ⊥ Department of Applied Chemistry, National Chiao Tung University, No. 1001, Ta-Hsueh Road, Hsinchu 300, Taiwan ‡

S Supporting Information *

ABSTRACT: Numerous biomolecules possess α-D-glucosamine as structural component. However, chemical glycosylations aimed at this backbone are usually not easily attained without generating the unwanted β-isomer. We report herein a versatile approach in affording full α-stereoselectivity built upon a carefully selected set of orthogonal protecting groups on a D-glucosaminyl donor. The excellent stereoselectivity provided by the protecting group combination was found independent of leaving groups and activators. With the trichloroacetimidate as the optimum donor leaving group, core skeletons of glycosylphosphatidyl inositol anchors, heparosan, heparan sulfate, and heparin were efficiently assembled. The orthogonal protecting groups were successfully manipulated to further carry out the total syntheses of heparosan tri- and pentasaccharides and heparin di-, tetra-, hexa-, and octasaccharide analogues. Using the heparin analogues, heparin-binding hemagglutinin, a virulence factor of Mycobacterium tuberculosis, was found to bind at least six sugar units with the interaction notably being entropically driven.

■

INTRODUCTION The numerous important roles of carbohydrates in living systems are underscored by their high abundance and molecular diversity. Once regarded as merely structural support and energy storage molecules, sugars are now acknowledged as vital players in physiological and pathophysiological processes making them potential drug development targets.1 They exist as glycoconjugates at the surface of every known cell, interacting and coordinating with various extracellular matrix elements.2 Several natural products also carry sugar moieties that influence their biological potency.3 With fewer monomer types than proteins, the exquisite complexity of carbohydrate polymers primarily arises from the multiple potential linkage points within the pyranosyl or furanosyl ring of each residue and the chirality of the anomeric center generated on assembly. Appreciation of the structure−function relationship of these biopolymers requires well-defined compounds, which are, unfortunately, difficult to isolate in sufficient quantity and purity from Nature. Thus, synthetic oligosaccharide preparations offer a dependable solution to vast demands.4 Several important biomolecules carry D-glucosamine in αform as core component. For example, glycosylphosphatidyl© 2012 American Chemical Society

inositol (GPI)-anchors, which tether proteins of various functions to the cell surface, contain D-glucosamine α1→6linked to myo-inositol (Figure 1).5 α-D-Glucosamine is also an

Figure 1. Structures of the α-D-glucosamine-containing disaccharide units found in GPI anchors, heparan sulfate, and heparin.

important feature, together with D-glucuronic and L-iduronic acid, in the repeating disaccharide of the structurally related heparan sulfate and heparin. Heparan sulfate is a linear polysulfated polysaccharide ubiquitously distributed on the Received: March 18, 2012 Published: May 15, 2012 8988

dx.doi.org/10.1021/ja302640p | J. Am. Chem. Soc. 2012, 134, 8988−8995

Journal of the American Chemical Society

Article

including heparosan and heparin, which were both synthesized in various lengths. With analogues of heparin at hand, we further examined their interaction with heparin-binding hemagglutinin (HBHA), a virulence factor of Mycobacterium tuberculosis.25

cell surface as a component of proteoglycans. It is a known mediator of several biologically important events including viral and bacterial infection, cell growth, inflammation, wound healing, tumor metastasis, lipid metabolism, and diseases of the nervous system.6 Found mainly in mast cells, heparin is a widely used clinical anticoagulant through its tight binding and consequent activation of antithrombin III.7 On the basis of this interaction, the synthetic pentasaccharide fondaparinux is now effectively utilized for the prevention of venous thrombolitic events following surgery.8 Alternating N-acetyl-α-D-glucosamine (GlcNAcα) and β-D-glucuronic acid all with 1→4 linkages are present in the capsular polysaccharide of Escherichia coli strain K5.9 Termed heparosan, it is actively being employed in the preparation of the more complex heparan sulfate/heparin saccharides through chemoenzymatic transformations.10 The highly immunogenic O-antigen of several serotypes of Shigella contains GlcNAcα in their repeating structure.11 GlcNAcα is also present in the cell-wall teichoic acid of some Staphylococcus species.12 Aside from these biopolymers, α-D-glucosamine is found in several natural products such as antibiotics (e.g., tunicamycins and neomycin)13 and bacterial antioxidants (e.g., mycothiol and bacillithiol).14 Because of the 1,2-cis nature of the α-D-glucosamine glycosidic bond, control of its stereoselective formation without the β-isomer is challenging.15 Typically, such couplings take advantage of the anomeric effectthe primary requirement of which is a nonparticipating group masking the 2-amine moiety. For this purpose, 2,3-trans-oxazolidinone, p-methoxybenzylideneamino group, and azide are the most prominent in recent literature. Initially applied by Kerns to D-glucosamine in 2001,16 the ring-fused oxazolidinone provided good α-stereoselectivity; but its use was curtailed by high promoter loading and several side reactions. Later modifications by acetylation17 or benzylation18 at the nitrogen position, together with the proper selection of activators and solvent, gave better stereocontrol and yield. Nguyen and co-workers, on the other hand, found that nickel catalysts, through coordination with the approaching acceptor and the p-methoxybenzylideneamino group at the 2-C position, could deliver the stereoselective α-glycosidation of trichloroacetimidate donors.19 The most commonly used 2amino protecting group is the azide, first utilized for its nonparticipating property in glycosylation by Paulsen20 more than 30 years ago. Nevertheless, it provided varying levels of αstereoselectivity, often rendering anomeric mixtures that led to tedious purification. Drawing from the observation that the axially oriented 4-hydroxyl of L-iduronate acceptors provided full α-stereoselectivity on glycosylation with 2-azido-2-deoxy-Dglucosyl donors, Seeberger suggested that forcing the hydroxyl nucleophile of the acceptor to assume the axial position would enable excellent stereoselectivity.21 Glycosylations of inositoltype acceptors following this concept, however, did not lead to the same outcome.22 Alternatively, Boons and co-workers reported that an added thioether to the reaction mixture assists in the α-selective glycosidation of D-glucosaminyl trichloroacetimidate donors.23 Our work on heparin and heparan sulfate synthesis24 elevated the desire to find a highly useful strategy for stereoselective α-glucosaminylation. Accordingly, we present herein a versatile approach in generating the α-glycosidic bond built upon a carefully selected set of orthogonal protecting groups on a D-glucosaminyl donor. Using this donor, backbones of some naturally occurring sugars were efficiently generated

■

RESULTS AND DISCUSSION α-Stereoselective Glucosaminylation. Besides the familiar 2-C neighboring group assistance, other functional groups also influence the stereoselectivity of glycosylation. For example, remote acyl moieties, bulky substituents, and fused diol protecting groups all demonstrated potent stereodirecting effects in a variety of glycosyl donors.26 Because the Dglucosamine residue of heparin/heparan sulfateour motivation for this explorationmay be modified at the 2-N, 3-O, and 6-O positions and must be amenable to further coupling at 4-O, some degree of orthogonality among the protecting groups is needed. For the 2-C functionality, we chose the azido group for it imparts low steric hindrance and does not complicate NMR analysis of sugar derivatives.27 It is also stable in many synthetic transformations, but is readily converted to the amine by reduction (e.g., hydrogenation or Staudinger reaction)28 or directly to the acetamide by thioacetic acid treatment,29 properties that nicely fit the D-glucosamine residues found in Nature. At 4-O, we required a blocking group that may both be a temporary and permanent protection. To this end, the 2naphthylmethyl (2-NAP) group is suitable because it can be chemoselectively cleaved by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or simultaneously removed together with the other permanent arylmethyl groups by hydrogenolysis in later synthetic steps. With these primary requirements aside, we examined the effect of several protecting groups at the 3-O and 6-O positions on the stereoselectivity of glucosaminylation in CH2Cl2 using the trichloroacetimidate as leaving group and the known 1,6-anhydro-β-L-idopyranosyl 4-alcohol 824f as glycosyl acceptor (Table 1). The α-products of such couplings possess the backbone of the major repeating component of heparin. Employing catalytic trimethylsilyl triflate (TMSOTf) as promoter, the markedly higher yield and α/β ratio were notable when benzoyl (Bz) group was utilized instead of benzyl (Bn) (entries 1−4). This increase in stereoselectivity probably resulted from the remote assistance by the Bz group. The installation of tert-butyldiphenylsilyl (TBDPS) moiety at the 6O position further gave better results (entries 5 and 6). Finally, when the p-bromobenzyl (p-BrBn) group was positioned at 3O while TBDPS blocks 6-O, complete stereoselectivity was afforded generating the α-disaccharide 15 in 84% yield (entry 7). Both situated at the β-face of the donor, these large groups may have prevented the approach of the acceptor at the zone that would generate the unwanted stereoisomer. Notably, methods are available for the chemoselective removal of TBDPS and p-BrBn groups,28b rendering donor 7 with a protecting group pattern that holds a high level of orthogonality. Following the successful stereoselective glycosylation of acceptor 8 using donor 7 and TMSOTf, other typical trichloroacetimidate promoters were further examined. The imidate activation by silver(I) triflate (AgOTf) and triflic acid (TfOH) exclusively furnished the α-adduct 15 in 63% (entry 8) and 66% (entry 9) yields, respectively. As for BF3·Et2O, the yield was a meager 26%; 49% of 8 was recovered (entry 10). We then focused on the effect of various leaving groups on the stereoselectivity of the 4-alcohol 8 glycosylation. The results 8989



Article

assembly of some important sugar skeletons (Scheme 1). Glycosylation of the myo-inositol 6-alcohol 2132 afforded the α-

Table 1. Coupling of the 4-Alcohol 8 and Variously Protected D-Glucosamine-Derived Trichloroacetimidate Donors

Scheme 1. Glycosylation of Various Acceptors Using the DGlucosaminyl Donor 7

entry

donor

promotera

time (h)

product (α/βb)

yield (%)

1 2 3 4 5 6 7 8c 9 10

1 2 3 4 5 6 7 7 7 7

TMSOTf TMSOTf TMSOTf TMSOTf TMSOTf TMSOTf TMSOTf AgOTf TfOH BF3·Et2O

1.5 1.5 1.5 1.5 2 2.5 2 2.5 2 1.5

9 (3.2/1) 10 (4.5/1) 11 (5.5/1) 12 (5.6/1) 13 (7.1/1) 14 (8.5/1) 15 (α only) 15 (α only) 15 (α only) 15 (α only)

63 71 72 73 89 76 84 63d 66d 26d

a

0.2 equiv TMSOTf, 5 equiv AgOTf, 0.2 equiv TfOH, and 1.2 equiv BF3·Et2O were used. bBased on isolated yields; the α-isomer has J1′,2′ of 3.7−3.8 Hz, whereas the β-isomer has 7.6−8.0 Hz. cThe reaction was carried out from −40 °C to rt. dRecovered acceptor 8: 13% (entry 8), 17% (entry 9), and 49% (entry 10). MS: molecular sieves.

pseudodisaccharide 22 in 71% yield as a single isomer. Compound 22 holds the core skeleton found in GPI anchors. The respective couplings of 7 with the 4-alcohols 2324d and 2533 both led to the disaccharides 24 and 26, which are also component backbones of heparin and heparan sulfate, in complete α-stereoselectivity and satisfactory yields. Additionally, 26 may be directly used for the synthesis of heparosan oligosaccharides. The preparation of compound 26 deserves more comment. Here, we utilized a trichloroacetimidate donor, a thioglycoside acceptor, and AgOTf as promoter. We found that promoting the reaction using TMSOTf, TfOH, or BF3·Et2O furnished the desired product in poor yields (>13%), a likely consequence of the commonly observed sulfide aglycon transfer.34 Synthesis of Heparosan Oligosaccharides. The enzymatic degradation of the E. coli strain K5 capsular polysaccharide supplies heparosan oligomers of various lengths.10 However, the isolation of sufficiently pure compound of defined size from this mixture is very difficult. With compound 26 in hand, we could generate synthetic heparosan oligosaccharides with precise structure and molecular weight. Described in Scheme 2 is the preparation of heparosan tri- and pentasaccharides, which may both act as substrates in chemoenzymatic transformations toward heparin/heparan sulfate structures. Condensation of the thioglycoside 26 and the acceptor 2735 mediated by NIS and TMSOTf supplied the trisaccharide 28 in 86% yield. Here, the Bz group situated at the 2-O position of the donor assisted in generating the exclusive βlinkage (J1′,2′ = 8.6 Hz). Cleavage of 2-NAP in 28 by DDQ supplied the 4″-alcohol (78%), which upon further glycosylation by 26 may form the pentasaccharide skeleton 29. Nonetheless, the glycosylation yield employing NIS and TMSOTf was only 12%, possibly because of the trisaccharide 4″-O-TMS formation, which may hinder effective coupling.

of the reaction leading to the disaccharide 15 are outlined in Table 2. Dehydrative glycosylation30 (entry 1), glycosyl Table 2. Coupling of the D-Glucosaminyl Donors 16−20 and the 4-Alcohol 8

entry

donor

promoter

1 2 3 4 5

16 17 18 19 20

Me2S/Tf2O NIS/TfOH TMSOTf TMSOTf AgOTf

temp (°C) rt −78 −40 −40 −20

to to to to

−20 0 0 0

time (h)

yield (%)

16 4 2 3 2.5

42a 79 73 48a 30a

a

Recovered acceptor 8: 16% (entry 1), 11% (entry 4), and 23% (entry 5). NIS: N-iodosuccinimide.

phosphate (entry 4), and glycosyl chloride (entry 5) gave modest results whereas thioglycoside (entry 2) and N-phenyl trifluoroacetimidate31 (entry 3) provided good yields. In all these cases, precedence toward α-selectivity was observed. Thus, the importance of proper selection of protecting groups in achieving α-stereoselectivity is highlighted. Our protecting group combination exclusively afforded the α-isomer amidst the use of various leaving groups and promoters. After establishing the effectiveness of our system with the 4alcohol 8, the trichloroacetimidate 7 was utilized in the 8990



Article

Scheme 2. Synthesis of the Heparosan Tri- and Pentasaccharidea

a Reagents and conditions: (a) NIS, TMSOTf, 4 Å MS, CH2Cl2, −78 °C to rt, 3 h; 86%. (b) (1) DDQ, CH2Cl2/H2O = 18/1, 2 h; 78%; (2) 26, NIS, TfOH, 4 Å MS, CH2Cl2, −78 °C to rt, 2 h; 64%. (c) (1) AcSH, Pyr, CHCl3, 36 h; (2) NaOMe, MeOH, 16 h; 30: 74%, 31: 72%. (d) TEMPO, BAIB, CH2Cl2/H2O = 2/1, 2 h; 32: 90%, 33: 77%. (e) (1) TBAF, AcOH, THF, 40 °C, 3 d; (2) H2, Pd(OH)2/C, phosphate buffer (pH 7), 2 d; 34: 91%, 35: 95%. Pyr: pyridine; TBAF: tetra-n-butylammonium fluoride.

Scheme 3. Synthesis of Heparin Analoguesa

Reagents and conditions: (a) DDQ, CH2Cl2/H2O = 18/1, rt, 4 h; 36: 92%, 40: 93%. (b) Ac2O, cat. Cu(OTf)2, 0 °C; 37: 99%, 41: 98%. (c) (1) satd NH3, MeOH/THF = 1/4, 0 °C, 6 h; (2) CCl3CN, K2CO3; 38: 90%, 42: 85%. (d) cat. TMSOTf, 4 Å MS, CH2Cl2, −40 to 0 °C, 2 h; 39: 98%, 43: 82%, 44: 84%. (e) NaOMe, CH2Cl2/MeOH = 1/1; 45: 98%, 46: 98%, 47: 91%, 48: 92%. (f) TEMPO, BAIB, CH2Cl2, 24 h; 49: 93%, 50: 78%, 51: 86%. (g) (1) TBAF, AcOH, THF, rt, 5 h; 95%; (2) SO3·Et3N, DMF, 60 °C, 3 d; 92%. (h) (1) TBAF, THF, 50 °C, 24 h, then, LiOH, rt, 3 h; (2) SO3·Et3N, DMF, 60 °C, 3 d; 53: 75%, 54: 73%; 55: 62%. (i) (1) PMe3, NaOH, THF/H2O = 9/1, rt, 5 h; (2) SO3·Pyr, Et3N, Pyr/DMF = 3/1, rt, 24 h; 56: 86%, 57: 75%, 58: 79%; 59: 69%. (j) H2, Pd(OH)2/C, phosphate buffer (pH 7), rt, 2 d; 60: 94%, 61: 90%, 62: 94%, 63: 87%. a

8991



Article

the cyclic ester was identified through the correlation between the 2-H and 6-C of the internal L-iduronyl residue in the HMBC spectra (Figure 2) and corroborated by the relatively

Conversely, when TfOH was used in place of TMSOTf, the target compound 29 was isolated in 64% yield. After constructing the oligosaccharide skeletons, functional group transformations were next carried out to achieve the desired materials. First, the azido groups of the trisaccharide 28 and pentasaccharide 29 underwent conversion to the acetamide using thioacetic acid. Subsequent treatment with NaOMe in MeOH resulted to deacylation delivering compounds 30 (74% from 28) and 31 (72% from 29). The primary hydroxyl groups in 30 and 31 were next oxidized to the carboxyls by employing a catalytic amount of 2,2,6,6-tetramethyl-1-piperidinyloxyl free radical (TEMPO) with excess [bis(acetoxy)iodo]benzene (BAIB) as co-oxidant to afford the acids 32 and 33 in 90% and 77% yields, respectively. Desilylation followed by global hydrogenolysis provided the target compounds 34 (91%) and 35 (95%), the structures of which were verified using NMR and mass spectroscopy (see the Supporting Information). Synthesis of Heparin Analogues. Heparin, being more readily accessible, is often used as a model in probing the character of heparan sulfate-mediated interactions the cell surface. Indeed, over 100 heparin-binding proteins have been identified.36 The major repeating component of heparin, among 48 disaccharide possibilities, is the N- and 6-O-sulfonated α-Dglucosamine 1→4-linked to 2-O-sulfonated α-L-iduronic acid. Acquisition of specific structurally defined heparin oligosaccharides from natural sources is a technical hurdle. Consequently, carbohydrate chemists developed several methodologies for their chemical and chemoenzymatic preparations.24,37 Scheme 3 illustrates our synthesis of the di-, tetra-, hexa-, and octasaccharide analogues of heparin starting from the disaccharide 15. The 2-NAP group and the easily transformed anhydroidose moiety allow conversion into either a glycosyl acceptor or donor. Accordingly, DDQ treatment of the fully protected skeletons 15 and 39 gave the acceptors 36 (92%) and 40 (93%), respectively. On the other hand, copper(II) triflate [Cu(OTf)2]-mediated acetolysis38 of the same starting materials provided the diacetate 37 (99%) and the triacetate 41 (98%), which were both converted into the respective imidates 38 and 42 by successive exposures to a saturated NH3 solution in MeOH/THF and CCl3CN in the presence of K2CO3. With the use of the acceptors 36 and 40 and the donors 38 and 42, the assembly of the heparin backbones 39 (98%), 43 (82%), and 44 (84%) under TMSOTf activation was achieved in a convergent and highly stereoselective manner. The W-coupling between the anomeric proton and 3-H, the small-to-zero coupling constant for 1-H, and the lack of NOE correlation between 1-H and 5-H of the involved L-idose residue are evidence that the newly formed glycosidic bond is α-configured and primarily exists in 1C4 conformation. Here, we chose to keep the bicyclic skeleton of the reducing end sugar to eliminate the installation of a noncarbohydrate aglycone that often results in a mixture of isomers, causing purification problems. A possible advantage of a rigid ring system at the reducing end is the relative ease of co-crystallization with a binding protein due to the reduced motion of the terminal group. Compounds 15, 39, 43, and 44 were next subjected to multifunctional group transformations leading to the target heparin analogues. Deacylation was conveniently carried out under Zemplén conditions to give the alcohols 45−48 in excellent yields. TEMPO oxidation of 46−48 resulted to the lactones 49 (93%), 50 (78%), and 51 (86%). The presence of

Figure 2. The HMBC spectrum of compound 49 identifying the lactone structure.

high IR CO stretching band observed around 1790 cm−1. Desilylation of 45 was accomplished using TBAF/AcOH reagent system to furnish the 2,6′-diol (95%), which was then treated with SO3·Et3N to afford the disulfate 52 in 92% yield. For 49−51, the basic TBAF affected not only the removal of TBDPS groups, but also the lactone ring opening as observed using TLC. LiOH was subsequently added to the solution to ascertain the complete cleavage of the lactone. O-Sulfonation of the partially purified materials gave the polysulfated compounds 53 (75% from 49), 54 (73% from 50), and 55 (62% from 51). Here, O-sulfonations were verified by the downfield shifts (>0.5 ppm) in the resonances of the respective geminal protons. The smooth azido-to-amino conversion was successfully implemented using PMe3 in THF/H2O with added NaOH to avoid the interference of the free carboxylic acid during the course of the reaction.39 Treatment with SO3·Pyr resulted in the amine sulfonation delivering the products 56 (86% from 52), 57 (75% from 53), 58 (79% from 54), and 59 (69% from 55). While Osulfonation could be detected from 1H NMR downfield shifts, no such trend occurs during N-sulfonation. Thankfully, we found that this functionalization can be identified using the highly indicative downfield shifts (4−5 ppm) in the 13C resonance of the nitrogen-bonded carbon (Figure 3). We also figured, upon comparison with the spectra of 32 and 33, that N-acetylated and N-sulfonated glucosamine residues can be differentiated by inspection of their 13C chemical shifts. Finally, the compounds were subjected to hydrogenolysis to remove all arylmethyl groups and supply the heparin analogues 60, 61, 62, and 63 in 94%, 90%, 94%, and 87% yields, respectively. The structures of these final compounds were confirmed by NMR and mass spectroscopic analysis (see the Supporting Information). Interaction of the Heparin Analogues and HBHA60−199. The invasion of alveolar macrophages by M. tuberculosis has an outstanding role in the pathogenesis of tuberculosis, a devastating disease that leaves millions dead each year.40 Besides this interaction, the bacteria also infect respiratory epithelial cells initiated upon adherence to heparan sulfate proteoglycans. The responsible bacterial adhesin is HBHA, a 199-amino acid protein, the absence of which severely impairs the extrapulmonary dissemination of the pathogen.41 HBHA has a coiled coil N-terminal domain associated with the agglutination property of the protein and a lysine-rich Cterminal region predisposed for binding with the polyanionic sugars of epithelial cells.42 8992



Article

Figure 3. Comparisons of carbons attached to azido, amino, sulfonatamido, and acetamido groups in DEPT-135 spectra (solvent: CD3OD).

We examined the interaction of the heparin analogues 60− 63 and a truncated form of HBHA (amino acid 60−199) using isothermal titration calorimetry (ITC) (Table 3). A commerTable 3. Thermodynamic Parameters of Sugar-Binding with HBHA60−199 Obtained Using ITCa sugar 63d 63 62 61 60 3 kDa heparin

nb 1.0 1.0 (±0.1) 1.1 (±0.2) −e −e 1.2 (±0.1)

KDc (μM)

ΔH (cal·mol−1)

ΔS (cal·mol−1·K−1)

ΔG (cal·mol−1)

19 4.2 (±0.4) 19 (±7)

3500 4370 (±150) 2400 (±90) − − 4220 (±1140)

33.3 39.3 (±0.3)

−6430 −7330

29.7 (±1.0)

−6450

− − 38.3 (±4.2)

− − −7200

− − 5.3 (±1.1)

Figure 4. Compound 63−HBHA60−199 ITC titration profile showing an endothermic interaction. Panel A shows the raw titration data and panel B shows the integrated heats of binding generated from the raw data after subtracting the heats of dilution.

for hexasaccharide 62. These data suggests a length of at least 6 sugar units for effective HBHA binding to occur.

■

CONCLUSIONS We have successfully developed a powerful donor for the formation of 2-deoxy-2-amino-α-D-glucosides in high stereoselectivity and good yields. The α-isomer was exclusively generated regardless of promoter and leaving group. Moreover, excellent α-stereoselectivities were afforded upon coupling with selected glycosyl acceptors to form the core structures of important sugars including GPI anchors, heparosan, heparan sulfate, and heparin. The orthogonal protecting groups built within our donor were effectively transformed and manipulated to furnish heparosan oligosaccharides and heparin analogues. Examination of the interaction of the prepared heparin analogues and HBHA60−199 revealed an entropically driven binding for the octasaccharide and hexasaccharide. Our disaccharide and tetrasaccharide heparin analogues did not bind to HBHA. From these examples, we have shown the flexibility of our donor in oligosaccharide synthesis. Further pursuit of other relevant bioactive compounds is currently in progress.

a

The values reported are averages of results from three experiments with standard deviations indicated in parentheses. bSugar-to-protein binding ratio. cThe actual parameter measured is association constant, the inverse of dissociation constant (KD). dThe protein utilized has an N-terminal hexahistidine tag; the values reported came from a single experiment. eNo binding was detected.

cially acquired 3 kDa heparin was also tested for comparison. Aggregation problems were incurred during the purification of the full-length HBHA expressed in E. coli; hence, we settled with its slightly shortened form. Moreover, the hexahistidine tag was cleaved from our recombinant protein prior to ITC measurement because it caused measurable decrease in binding affinity. Here, the protein solution in Tris buffer (pH 7.6) was titrated with the sugar dissolved in the same solvent at 25 °C. To account for the contribution of heat of dilution, the buffer without the protein was also titrated with the sugar solution. No HBHA60−199 binding was detected for the disaccharide 60 and tetrasaccharide 61. On the other hand, the associations of HBHA60−199 with the hexasaccharide 62, the octasaccharide 63, and the commercial heparin were all found to be endothermic with the sugar−protein binding stabilized by entropic factors. A representative titration isotherm is shown in Figure 4. The release of the ordered water molecules from the interface between HBHA60−199 and the sugar molecules are quite possibly the major driving force for the adhesion process. Compound 63 has a slightly better binding than the 3 kDa heparin with both sugars having nearly 1:1 binding stoichiometry with HBHA60−199. The binding affinity is lower

■

EXPERIMENTAL SECTION

General Procedure for the Synthesis of Compounds 9−15. A solution of the trichloroacetimidate donor (1−7, 1.2 equiv) and the 4alcohol 8 (1.0 equiv) in CH2Cl2 (10 mL per total gram of donor, acceptor, and molecular sieves) with freshly dried 4 Å molecular sieves (1.5 g per total gram of the donor and the acceptor) was stirred at room temperature for 1 h under N2 atmosphere. The mixture was cooled to −78 °C, TMSOTf (0.2 equiv) was added to the solution, and the mixture was gradually warmed up to 0 °C. After stirring for 1.5−2.5 h, Et3N (0.3 equiv) was added to quench the reaction and the 8993



Article

mixture was filtered through Celite. The filtrate was concentrated under reduced pressure to furnish a residue, which was purified by flash column chromatography to give the expected disaccharide. The yields of compounds 9−15 are listed in Table 1.

■

(13) (a) Kimura, K.-i.; Bugg, T. D. H. Nat. Prod. Rep. 2003, 20, 252− 273. (b) Magnet, S.; Blanchard, J. S. Chem. Rev. 2005, 105, 477−497. (14) (a) Newton, G. L.; Buchmeier, N.; Fahey, R. C. Microbiol. Mol. Biol. Rev. 2008, 72, 471−494. (b) Helmann, J. D. Antioxid. Redox Signaling 2011, 15, 123−133. (15) Bongat, A. F. G.; Demchenko, A. V. Carbohydr. Res. 2007, 342, 374−406. (16) (a) Benakli, K.; Zha, C.; Kerns, R. J. J. Am. Chem. Soc. 2001, 123, 9461−9462. (b) Kerns, R. J.; Zha, C.; Benakli, K.; Liang, Y.-Z. Tetrahedron Lett. 2003, 44, 8069−8072. (17) (a) Wei, P.; Kerns, R. J. J. Org. Chem. 2005, 70, 4195−4198. (b) Boysen, M.; Gemma, E.; Lahmann, M.; Oscarson, S. Chem. Commun. 2005, 3044−3046. (c) Geng, Y.; Zhang, L.-H.; Ye, X.-S. Chem. Commun. 2008, 597−599. (18) (a) Manabe, S.; Ishii, K.; Ito, Y. J. Am. Chem. Soc. 2006, 128, 10666−10667. (b) Manabe, S.; Ishii, K.; Ito, Y. Eur. J. Org. Chem. 2011, 497−516. (19) (a) Mensah, E. A.; Nguyen, H. M. J. Am. Chem. Soc. 2009, 131, 8778−8780. (b) Mensah, E. A.; Yu, F.; Nguyen, H. M. J. Am. Chem. Soc. 2010, 132, 14288−14302. (20) Paulsen, H.; Kolár,̌ Č .; Stenzel, W. Angew. Chem., Int. Ed. Engl. 1976, 15, 440−441. (21) Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Seeberger, P. H. Angew. Chem., Int. Ed. 2002, 41, 2128−2131. (22) Cid, M. B.; Alfonso, F.; Martín-Lomas, M. Chem.Eur. J. 2005, 11, 928−938. (23) Park, J.; Kawatkar, S.; Kim, J.-H.; Boons, G.-J. Org. Lett. 2007, 9, 1959−1962. (24) (a) Hung, S.-C.; Thopate, S. R.; Chi, F.-C.; Chang, S.-W.; Lee, J.-C.; Wang, C.-C.; Wen, Y.-S. J. Am. Chem. Soc. 2001, 123, 3153− 3154. (b) Lee, J.-C.; Chang, S.-W.; Liao, C.-C.; Chi, F.-C.; Chen, C.-S.; Wen, Y.-S.; Wang, C.-C.; Kulkarni, S. S.; Puranik, R.; Liu, Y.-H.; Hung, S.-C. Chem.Eur. J. 2004, 10, 399−415. (c) Lee, J.-C.; Lu, X.-A.; Kulkarni, S. S.; Wen, Y.-S.; Hung, S.-C. J. Am. Chem. Soc. 2004, 126, 476−477. (d) Lu, L.-D.; Shie, C.-R.; Kulkarni, S. S.; Pan, G.-R.; Lu, X.A.; Hung, S.-C. Org. Lett. 2006, 8, 5995−5998. (e) Hu, Y.-P.; Lin, S.Y.; Huang, C.-Y.; Zulueta, M. M. L.; Liu, J.-Y.; Chang, W.; Hung, S.-C. Nat. Chem. 2011, 3, 557−563. (f) Hung, S.-C.; Lu, X.-A.; Lee, J.-C.; Chang, M. D.-T.; Fang, S.-l.; Fan, T.-c.; Zulueta, M. M. L.; Zhong, Y.Q. Org. Biomol. Chem. 2012, 10, 760−772. (25) Locht, C.; Hougardy, J.-M.; Rouanet, C.; Place, S.; Mascart, F. Tuberculosis 2006, 86, 303−309. (26) (a) Lipták, A; Borbás, A.; Bajza, I. In Comprehensive Glycoscience: From Chemistry to Systems Biology; Kamerling, J. P., Ed.; Elsevier: Amsterdam, 2007; pp 203−259. (b) Codée, J. D. C.; Ali, A.; Overkleeft, H. S.; van der Marel, G. A. C. R. Chim. 2011, 14, 178−193. (27) Nyffeler, P. T.; Liang, C.-H.; Koeller, K. M.; Wong, C.-H. J. Am. Chem. Soc. 2002, 124, 10773−10778. (28) (a) Scriven, E. F. V.; Turnbull, K. Chem. Rev. 1988, 88, 297− 368. (b) Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in Organic Synthesis, 4th ed; John Wiley & Sons, Inc.: Hoboken, NJ, 2007. (29) (a) Rosen, T.; Lico, I. M.; Chu, D. T. W. J. Org. Chem. 1988, 53, 1580−1582. (b) Shangguan, N.; Katukojvala, S.; Greenberg, R.; Williams, L. J. J. Am. Chem. Soc. 2003, 125, 7754−7755. (30) Nguyen, H. M.; Chen, Y.; Duron, S. G.; Gin, D. Y. J. Am. Chem. Soc. 2001, 123, 8766−8772. (31) Yu, B.; Tao, H. Tetrahedron Lett. 2001, 42, 2405−2407. (32) Patil, P. S.; Hung, S.-C. Chem.Eur. J. 2009, 15, 1091−1094. (33) Prepared according to our regioselective one-pot protection strategy: (a) Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Kulkarni, S. S.; Huang, Y.-W.; Lee, C.-C.; Chang, K.-L.; Hung, S.-C. Nature 2007, 446, 896− 899. (b) Wang, C.-C.; Kulkarni, S. S.; Lee, J.-C.; Luo, S.-Y.; Hung, S.C. Nat. Protoc. 2008, 3, 97−113. (c) Chang, K.-L.; Zulueta, M. M. L.; Lu, X.-A.; Zhong, Y.-Q.; Hung, S.-C. J. Org. Chem. 2010, 75, 7424− 7427. (34) Li, Z.; Gildersleeve, J. C. J. Am. Chem. Soc. 2006, 128, 11612− 11619.

ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental procedures and 1H and 13C NMR spectra for relevant compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

[email protected]; [email protected] Author Contributions #

These authors contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We dedicate this paper to Prof. Chun-Chen Liao on the occasion of his 70th birthday. This work was supported by the National Science Council (NSC 97-2113-M-001-033-MY3, NSC 98-2119-M-001-008-MY2) and Academia Sinica. The authors thank Dr. Wei-Juin Su for providing the DNA of HBHA from M. tuberculosis H37Rv.

■

REFERENCES

(1) (a) Varki, A. Glycobiology 1993, 3, 97−130. (b) Dwek, R. Chem. Rev. 1996, 96, 683−720. (c) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357−2364. (d) Ernst, B.; Magnani, J. L. Nat. Rev. Drug Discovery 2009, 8, 661−677. (2) (a) Collins, B. E.; Paulson, J. C. Curr. Opin. Chem. Biol. 2004, 8, 617−625. (b) Mager, M. D.; LaPointe, V.; Stevens, M. M. Nat. Chem. 2011, 3, 582−589. (3) Thorson, J. S.; Vogt, T. In Carbohydrate-Based Drug Discovery; Wong, C.-H., Ed.; Wiley-VCH: Weinheim, 2003; pp 685−711. (4) (a) Boltje, T. J.; Buskas, T.; Boons, G.-J. Nat. Chem. 2009, 1, 611−622. (b) Galan, M. C.; Benito-Alifonso, D.; Watt, G. M. Org. Biomol. Chem. 2011, 9, 3598−3610. (c) Hsu, C.-H.; Hung, S.-C.; Wu, C.-Y.; Wong, C.-H. Angew. Chem., Int. Ed. 2011, 50, 11872−11923. (5) Ferguson, M. A. J.; Williams, A. F. Annu. Rev. Biochem. 1988, 57, 285−320. (6) (a) Whitelock, J. M.; Iozzo, R. V. Chem. Rev. 2005, 105, 2745− 2764. (b) Bishop, J. R.; Schuksz, M.; Esko, J. D. Nature 2007, 446, 1030−1037. (7) Linhardt, R. J. J. Med. Chem. 2003, 46, 2551−2564. (8) Petitou, M.; van Boeckel, C. A. A. Angew. Chem., Int. Ed. 2004, 43, 3118−3133. (9) Vann, W. F.; Schmidt, M. A.; Jann, B.; Jann, K. Eur. J. Biochem. 1981, 116, 359−364. (10) (a) Lindahl, U.; Li, J.-p.; Kusche-Gullberg, M.; Salmivirta, M.; Alaranta, S.; Veromaa, T.; Emeis, J.; Roberts, I.; Taylor, C.; Oreste, P.; Zoppetti, G.; Naggi, A.; Torri, G.; Casu, B. J. Med. Chem. 2005, 48, 349−352. (b) Muñoz, E. M.; Xu, D.; Avci, F. Y.; Kemp, M.; Liu, J.; Linhardt, R. J. Biochem. Biophys. Res. Commun. 2006, 339, 597−602. (c) Chen, J.; Jones, C. L.; Liu, J. Chem. Biol. 2007, 14, 986−993. (d) Peterson, S.; Frick, A.; Liu, J. Nat. Prod. Rep. 2009, 26, 610−627. (11) Liu, B.; Knirel, Y. A.; Feng, L.; Perepelov, A. V.; Senchenkova, S. N.; Wang, Q.; Reeves, P. R.; Wang, L. FEMS Microbiol. Rev. 2008, 32, 627−653. (12) (a) Sadovskaya, I.; Vinogradov, E.; Li, J.; Jabbouri, S. Carbohydr. Res. 2004, 339, 1467−1473. (b) Xia, G.; Maier, L.; Sanchez-Carballo, P.; Li, M.; Otto, M.; Holst, O.; Peschel, A. J. Biol. Chem. 2010, 285, 13405−13415. 8994



Article

(35) Shie, C.-R.; Tzeng, Z.-H.; Kulkarni, S. S.; Uang, B.-J.; Hsu, C.-Y.; Hung, S.-C. Angew. Chem., Int. Ed. 2005, 44, 1665−1668. (36) (a) Conrad, H. E. Heparin-Binding Proteins; Academic Press: San Diego, CA, 1998. (b) Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002, 41, 390−412. (37) (a) Noti, C.; Seeberger, P. H. Chem. Biol. 2005, 12, 731−756. (b) Noti, C.; de Paz, J. L.; Polito, L.; Seeberger, P. H. Chem.Eur. J. 2006, 12, 8664−8686. (c) Polat, T.; Wong, C.-H. J. Am. Chem. Soc. 2007, 129, 12795−12800. (d) Adibekian, A.; Bindschädler, P.; Timmer, M. S. M.; Noti, C.; Schützenmeister, N.; Seeberger, P. H. Chem.Eur. J. 2007, 13, 4510−4522. (e) Arungundram, S.; AlMafraji, K.; Asong, J.; Leach, F. E., III; Amster, I. J.; Venot, A.; Turnbull, J. E.; Boons, G.-J. J. Am. Chem. Soc. 2009, 131, 17394− 17405. (f) Baleux, F.; Loureiro-Morais, L.; Hersant, Y.; Clayette, P.; Arenzana-Seisdedos, F.; Bonnaffé, D.; Lortat-Jacob, H. Nat. Chem. Biol. 2009, 5, 743−748. (g) Liu, R.; Xu, Y.; Chen, M.; Weïwer, M.; Zhou, X.; Bridges, A. S.; DeAngelis, P. L.; Zhang, Q.; Linhardt, R. J.; Liu, J. J. Biol. Chem. 2010, 285, 34240−34249. (h) Wang, Z.; Xu, Y.; Yang, B.; Tiruchinapally, G.; Sun, B.; Liu, R.; Dulaney, S.; Liu, J.; Huang, X. Chem.Eur. J. 2010, 16, 8365−8375. (i) Tiruchinapally, G.; Yin, Z.; El-Dakdouki, M.; Wang, Z.; Huang, X. Chem.Eur. J. 2011, 17, 10106−10112. (j) Xu, Y.; Masuko, S.; Takieddin, M.; Xu, H.; Liu, R.; Jing, J.; Mousa, S. A.; Linhardt, R. J.; Liu, J. Science 2011, 334, 498− 501. (38) Kulkarni, S. S.; Hung, S.-C. Lett. Org. Chem. 2005, 2, 670−677. (39) Brewer, M.; Rich, D. H. Org. Lett. 2001, 3, 945−948. (40) Russel, D. G. Immunol. Rev. 2011, 240, 252−268. (41) Pethe, K.; Alonso, S.; Biet, F.; Delogu, G.; Brennan, M. J.; Locht, C.; Menozzi, F. D. Nature 2001, 412, 190−194. (42) Esposito, C.; Pethoukov, M. V.; Svergun, D. I.; Ruggiero, A.; Pedone, C.; Pedone, E.; Berisio, R. J. Bacteriology 2008, 190, 4749− 4753.

8995


Î±-Glycosylation by d-Glucosamine-Derived Donors: Synthesis of

Recommend Documents